Compositions and methods for enhancing exercise performance

ABSTRACT

Compositions and methods are provided for improving canine exercise performance. The compositions are pre-exercise supplements that generally comprise (a) about 35% to about 60% protein or amino acids, comprising one or more structural proteins, one or more bioavailable proteins and one or more branched chain amino acids; (b) about 20% to about 38% fat, comprising at least one source of medium chain triglycerides; and (c) about 5% to about 25% carbohydrate. The methods involve administering the supplement to the animal within about 30-60 minutes before the beginning of the exercise session. The supplements can be administered in conjunction with one or more other exercise performance-enhancing or recovery agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/310,359 filed Jun. 20, 2014 that claims priority to U.S. ProvisionalApplication Ser. No. 61/841,011 filed Jun. 28, 2013, the disclosures ofwhich are incorporated in their entirety herein by these references.

BACKGROUND OF THE INVENTION

Field of the Invention

The invention relates generally to field of exercise performance andnutrition in support thereof. In particular, the invention relates todietary supplements containing proteins and fats that are readilyabsorbed, and methods for administering the supplements within a definedperiod prior to exercise.

Description of Related Art

Performance improvement for canines can be related to increased staminaor endurance while running, tracking, retrieving, swimming, pulling, orany other activity that requires sustained physical movement or exertionduring a required task or activity. For dogs, fat metabolism is theprimary route for generating energy during sub-maximal exertion. Dogsare inherently “programmed” (evolved) for high endurance activity andmetabolism. However, depending on the level of exercise conditioning,physical fitness, and frequency of exercise, fatigue will occur. Hence,prolonging endurance and/or performance is of clear benefit, so havingan performance enhancing food product or nutrient delivery system wouldbe advantageous.

In dogs, fat oxidation provides most of the animal's energy at low ratesof energy expenditure, whereas with humans, energy is morepreferentially generated from glucose from glycogen stores. Morespecifically, for dogs, the amount of energy from fat oxidation at restand during exercise is twice that in less aerobic species such as goats(McLelland et al., 1994, Am. J. Physiol. 266 (4 Pt. 2): R1280-1286;Weibel et al., 1996, J. Exp. Biol. 199 (8): 1699-1709). In humans,carbohydrate oxidation supports the intermediate speed of marathonrunners until all glycogen stores have been depleted, whereupon fatoxidation becomes the only source available for energy (Hultman et al.,1994, in Modern Nutrition in Health and Disease, M. E. Shils, J. A.Olson, and M. Shike, eds; Lea & Febiger, Philadelphia, pp. 663-685).Consequently, stamina for humans is limited by the amount of glycogen inmuscle, whereas in dogs, activation of fat metabolism and conversion ofamino acids into glucose becomes initiated soon after exercise, thuscontributing to an overall increased aerobic capacity and endurance.

Muscle fiber variation among different animals also contributes to thedegree in which either aerobic or anaerobic sources of energy are used.In dogs and cats, muscles contain different muscle fiber types.Specifically, all fiber types in dogs have high aerobic capacity, thusare fatigue resistant. In contrast, the muscle fibers of cats can bedivided into low aerobic type that relies on anaerobic metabolism, or ahigh aerobic type with high capacity for aerobic metabolism.Consequently, maximum oxygen metabolism capacity (V_(O2)max) and bloodflow at V_(O2)max in leg (gastrocnemius) muscles have been determined tobe about five times higher in dogs than cats. Thus, dogs are adapted forendurance exercise using fat as an energy source, whereas cats areadapted for short bursts of activity such as that required when jumpingand pouncing on prey, thus using glycogen as the energy source.

Exercise performance for canines is also significantly related toprotein and amino acid metabolism. Protein and amino acid synthesis andcatabolism increase in exercising dogs. Synthesis increases toaccommodate the changes associated with training and to replace proteinand amino acids catabolized during exercise. Protein and amino acids arecatabolized during exercise as a source of energy, particularly withinexercising muscles, and as precursors of gluconeogenesis.Gluconeogenesis plays an important role in exercise, particularly inexercise lasting longer than 30 minutes, as gluconeogenic precursorslike alanine, lactate, pyruvate and glutamine increase. Importantly,precursors for gluconeogenesis are mobilized from the muscle, gut, andadipose tissue. Glutamine and alanine are important gluconeogenicprecursors because they shuttle the ammonia by-product resulting frombranched-chain amino acid oxidation out of exercising muscles forconversion to glucose by the liver. Consequently, labile protein sourcesin the gut appear to be the source of branched-chain amino acids thatsupport exercising muscles and are liberated as a result ofexercise-induced muscle protein catabolism.

Physical performance can become altered as a result of cell stress andcellular damage to the muscle. Cellular damage is a natural consequenceof exercise, and results from excessive protein catabolism, as well asoxidative stress occurring from free-radicals generated by aerobicrespiration. The term “cellular damage” is well established within thesport/exercise research community. The degree to which this metaboliccondition occurs is related to several factors, including conditioning,duration, intensity, and recurrence of exercise, as well as tonutrition, which can be used to alter this metabolic condition in afavorable manner.

Following exercise-induced muscle damage there is a reduction in theability of the muscle to contract with maximal force (Pearce et al.,1998, J. Sci. Med. Sport 1: 236-244), which is observed in all threetypes of muscle. action; eccentric (lowering of lifted weight orlengthening of muscle), concentric (lifting weight or shortening ofmuscle), and isometric (muscle length does not change duringcontraction) (Turner et al., 2008, J. Appl. Physiol. 105: 502-509), aswell as jumping (Kirby et al., 2012, Amino Acids 42(5): 1987-1996).

The depletion of intracellular branched-chain amino acid levels inmuscle cells during exercise results from their oxidation as an energysource. Intracellular depletion of branched-chain amino acids,particularly leucine, also corresponds to a reduction in circulatingplasma concentrations. Doses of BCAAs (0.050 to 0.100 g/kg BW) ingestedbefore endurance and resistance exercise reduced muscle damagebiomarkers (i.e., CK), muscle soreness during recovery (DOMS: delayedonset of muscle soreness) and reduced muscle fatigue (Greer et al.,2007, Int. J. Sport Nutr. Exerc. Metab. 17(6): 595-607; Jackman et al.,2010, Med. Sci. sports Exerc. 42(5): 962-70).

Currently, there is no effective food for animals, particularly fordogs, that can be fed before exercise that can adequately prime theirmetabolism or minimize endogenous protein breakdown to enhance theirendurance and/or reduce the natural catabolic state induced by exercise,and subsequently improve their performance during an exercise bout.Furthermore, reducing the extent of the exercise-induced catabolic statewill hasten post-exercise recovery. For example, within the dogperformance snack market, current products are formulated with highlevels of carbohydrates and very little fat—likely below 6%. This wouldlead to an elevation in insulin secretion, which would result in areduction in the activity of exercise-related metabolic pathways.Typically, these products also are low in protein (e.g., below 10%) andthe protein sources they contain are not formulated for ready digestion.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to providecompositions and methods useful for enhancing exercise performance in ananimal, particularly a canine.

It is another object of the present invention to provide compositionsand methods useful for enhancing exercise performance in an animal,particularly a canine, by increasing the bioavailability of dietaryprotein in the animal.

It is another object of the present invention to provide compositionsand methods useful for enhancing exercise performance in an animal,particularly a canine, by increasing the bioavailability ofbranched-chain amino acids in the animal.

It is another object of the present invention to provide compositionsand methods useful for enhancing exercise performance in an animal,particularly a canine, by increasing the bioavailability of leucine inthe animal

It is another object of the present invention to provide compositionsand methods useful for enhancing exercise performance in an animal,particularly a canine, by reducing the oxidation of endogenous sourcesof branched-chain amino acids in the animal.

It is another object of the present invention to provide compositionsand methods useful for enhancing exercise performance in an animal,particularly a canine, by activating protein synthesis in the animal.

It is a further object of the invention to provide kits for improvingexercise performance in the animal, and for use in the methods describedherein.

It is another object of the invention to provide a package comprising acomposition provided herein and a label, logo, graphic or the likeaffixed to the package indicating the contents of the package and/or thebenefits of administering the dietary supplements to an animal for thepurposes of improving exercise performance.

One or more of these and other objects are achieved using novelcompositions and methods for enhancing exercise performance in animals.Generally, the compositions can comprise a pre-exercise supplement thatincludes (a) about 35% to about 60% protein or amino acids, comprisingone or more structural proteins, one or more bioavailable proteins andone or more branched chain amino acids; (b) about 20% to about 38% fat,comprising at least one source of medium chain triglycerides; and (c)about 5% to about 25% carbohydrate. The methods comprise administeringthe supplement within a pre-determined period prior to the commencementof exercise.

These and other and further objects, features, and advantages of thepresent invention will be readily apparent to those skilled in the art.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “individual” when referring to an animal means an individualanimal of any species or kind, most particularly a canine.

The term “animal” means any animal that could benefit from one or moreof the compositions and methods of the provided herein, particularly ananimal that could benefit from methods and compositions that are usefulfor enhancing exercise performance. Thus, the instant disclosure relatesto any animal. In one embodiment, the animal can be a canine. In oneaspect, the canine can be a domesticated species, such as a dog. Forexample, certain canine companions are subjected to physical activitythat can be strenuous, particularly canines that are used for work, suchas sledding or carting, herding, police work, rescue, tracking, sportand agility. Alternatively, the canine can be a semi-domesticated orundomesticated species, such as a dingo, wolf, coyote, jackal or fox.

As used herein “exercise” is a type of physical activity undertaken byan animal or caused to be undertaken by an animal for a particularpurpose such as general heath, fitness, weight management, improving aparticular aspect of health or fitness, strengthening, improving aphysical skill or set of skills, improving a function, rehabilitating aninjury, and the like. Exercise can be performed on a regular basis, forexample daily, thrice per week, or once per week. Frequencies ofexercise less than once per week are considered “occasional” exercise.Other patterns of exercise are also recognized and contemplated for useherein. The compositions and methods are useful with exercise whetherregular or occasional.

The term “effective amount” means an amount of a compound, material,composition, medicament, or other material that is effective to achievea particular biological result. Such results include, but are notlimited to, one or more of the following outcomes set forth hereinbelow.

The term “supplement” or “dietary supplement” means a product that isintended to be ingested in addition to the normal animal diet. Dietarysupplements may be in any form, e.g., solid, liquid, gel, tablets,capsules, powder, and the like. In one embodiment, they are provided inconvenient dosage forms. In some embodiments they are provided in bulkconsumer packages such as bulk powders, liquids, gels, or oils. In otherembodiments, supplements are formulated as food items such as snacks,treats, biscuits, beverages and the like.

The terms “administering” or “administration” includeself-administration in addition to administration to another animal, forexample a caretaker may administer a dietary supplement to a companionanimal.

The term “oral administration” or “orally administering” means that theanimal ingests, or a human is directed to feed, or does feed, the animalone or more of the compositions described herein. Wherein a human isdirected to feed the composition, such direction may be that whichinstructs and/or informs the human that use of the composition mayand/or will provide the referenced benefit, for example, enhancingperformance during physical activity or exercise. Such direction may beoral direction (e.g., through oral instruction from, for example, aphysician, veterinarian, or other health professional, or radio ortelevision media (i.e., advertisement), or written direction (e.g.,through written direction from, for example, a physician, veterinarian,or other health professional (e.g., prescriptions), sales professionalor organization (e.g., through, for example, marketing brochures,pamphlets, or other instructive paraphernalia), written media (e.g.,internet, electronic mail, website, or other computer-related media),and/or packaging associated with the composition (e.g., a label presenton a container holding the composition), or a combination thereof (e.g.,label or package insert with directions to access a website for moreinformation).

The term “in conjunction” means that a composition for improvingexercise performance, a food composition, medicament, drug, recoveryagent, or other compound or composition described herein areadministered to an animal (1) together in a single composition or (2)separately at the same or different frequency using the same ordifferent administration routes at about the same time or periodically.“Periodically” means that the agent is administered on a dosage scheduleacceptable for a specific agent and that the food or supplement is fedto an animal routinely as appropriate for the particular animal. “Aboutthe same time” generally means that the supplement and agent areadministered at the same time or within about 72 hours of each other.“In conjunction” specifically includes administration schemes wherein adietary supplement as disclosed herein is administered within a definedwindow of time before exercise, the window being between about 0-120minutes before the start of the exercise.

The term “single package” means that the components of a kit arephysically associated, in or with one or more containers, and considereda unit for manufacture, distribution, sale, or use. Containers include,but are not limited to, bags, boxes or cartons, bottles, packages of anytype or design or material, over-wrap, shrink-wrap, affixed components(e.g., stapled, adhered, or the like), or combinations of any of theforegoing. For example, a single package kit may provide containers ofindividual compositions and/or food compositions physically associatedsuch that they are considered a unit for manufacture, distribution,sale, or use.

The term “virtual package” means that the components of a kit areassociated by directions on one or more physical or virtual kitcomponents instructing the user how to obtain the other components,e.g., in a bag or other container containing one component anddirections instructing the user to go to a website, contact a recordedmessage or a fax-back service, view a visual message, or contact acaregiver or instructor to obtain, for example, instructions on how touse the kit, or safety or technical information about one or morecomponents of a kit. Examples of information that can be provided aspart of a virtual kit include instructions for use; safety informationsuch as material safety data sheets; poison control information;information on potential adverse reactions; clinical study results;dietary information such as food composition or caloric composition;general information on physical activity, exercise, metabolism,endurance and the like.

All percentages expressed herein are by weight of the composition on adry matter basis unless specifically stated otherwise. The skilledartisan will appreciate that the term “dry matter basis” means that aningredient's concentration or percentage in a composition is measured ordetermined after any free moisture in the composition has been removed.

Ranges are used herein in shorthand, so as to avoid having to set out atlength and describe each and every value within the range. Anyappropriate value within the range can be selected, where appropriate,as the upper value, lower value, or the terminus of the range.

Where used herein, the term “about” indicates that the given value, plusor minus 20% or 15% or 10% or 5% or 1%, is intended. “About” is thusused a shorthand to reflect the recognition that small variations fromthe literal value stated are still within the scope of the invention.

As used herein and in the appended claims, the singular form of a wordincludes the plural, and vice versa, unless the context clearly dictatesotherwise. Thus, the references “a”, “an”, and “the” are generallyinclusive of the plurals of the respective terms. For example, referenceto “a dog”, “a method”, or “a supplement” includes a plurality of such“dogs”, “methods”, or “supplements”. Similarly, the words “comprise”,“comprises”, and “comprising” are to be interpreted inclusively ratherthan exclusively. Likewise the terms “include”, “including” and “or”should all be construed to be inclusive, unless such a construction isclearly prohibited from the context. Where used herein “examples,” or“for example,” particularly when followed by a listing of terms, ismerely exemplary and illustrative, and should not be deemed to beexclusive or comprehensive.

The term “comprising” is intended to include embodiments encompassed bythe terms “consisting essentially of” and “consisting of”. Similarly,the term “consisting essentially of” is intended to include embodimentsencompassed by the term “consisting of”.

The methods and compositions and other advances disclosed here are notlimited to particular methodology, protocols, and reagents describedherein because they may be varied in ways that are apparent the skilledartisan. Further, the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to, and doesnot, limit the scope of that which is disclosed or claimed.

Unless defined otherwise, all technical and scientific terms, terms ofart, and acronyms used herein have the meanings commonly understood byone of ordinary skill in the art in the field(s) of the invention, or inthe field(s) where the term is used. Although any compositions, methods,articles of manufacture, or other means or materials similar orequivalent to those described herein can be used in the practice of thepresent invention, certain compositions, methods, articles ofmanufacture, or other means or materials are described herein.

All patents, patent applications, publications, technical and/orscholarly articles, and other references cited or referred to herein arein their entirety incorporated herein by reference to the extent allowedby applicable law. The discussion of those references is intended merelyto summarize the assertions made therein. No admission is made that anysuch patents, patent applications, publications or references, or anyportion thereof, are relevant, material, or prior art. The right tochallenge the accuracy and pertinence of any assertion of such patents,patent applications, publications, and other references as relevant,material, or prior art is specifically reserved. Full citations forpublications not cited fully within the specification are set forth atthe end of the specification.

The Invention

The present inventors have recognized that most formulated energyproducts for dogs include a variety of vitamin/mineral and carbohydrateformulations touted to promote performance, but none target theunderlying physiology of enhancing protein building or reducing proteinbreakdown for exercise. In addition, specific feeding times forfoods/supplements before exercise have not been understood as impactingperformance. Accordingly, the present inventors have discovered newcompositions and methods that influence and improve exercise performancein an animal, particularly a canine.

One aspect of the invention features a pre-exercise dietary supplementfor an animal. In certain embodiments, the animal is one whosemetabolism uses fat as a first and/or primary energy source in enduranceexercise. In one embodiment, the animal is a canine.

The supplement can comprise: (a) about 35% to about 60% protein,peptides and/or amino acids, comprising one or more structural proteins,one or more bioavailable proteins and one or more branched chain aminoacids; (b) about 20% to about 38% fat, comprising at least one source ofmedium chain triglycerides; and (c) about 5% to about 25% carbohydrate,in one aspect, comprising complex carbohydrates.

In certain embodiments, the supplement comprises at least about 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58% or 59% protein, peptidesand/or amino acids. In certain embodiments, the supplement comprises upto about 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%protein, peptides and/or amino acids. In particular, the supplement cancomprise between about 40% and about 50% protein, peptides and/or aminoacids, or between about 45% and about 55% protein, peptides and/or aminoacids. In one embodiment, the protein/peptide/amino acid component ofthe supplement will include about 30-46% structural protein, about24-31% medium length peptide fragments, and about 20-50% bioavailableproteins/peptides. Distributed within the protein/peptide/amino acidcomponent are about 6-8% branched chain amino acids (the latterpercentage based on the total product).

As mentioned, the protein/peptide/amino acid components of thesupplement include structural protein, bioavailable proteins orpeptides, and amino acids. The structural protein functions to provideshape, texture and nutritional content to the supplement. Structuralproteins can include animal proteins such as muscle and organ protein.In one embodiment, the structural protein includes heart muscle, such asbeef heart, and may also include a vegetable protein such as soy meal,flour or grits.

The bioavailable protein or peptide component functions to provide arapidly absorbed protein source at the outset of the exercise period.Such rapidly absorbed protein sources can include those that areabsorbed within 30 minutes upon ingestion, and in one aspect, within 15minutes. Bioavailable proteins/peptides can include whey, partiallyhydrolyzed soy protein, hydrolyzed amino acids, or any combinationthereof. The whey protein, if present, comprises a whey proteinconcentrate with about 80% crude protein; the soy, if present is in theform of Soy Protein Modified (e.g., SPI 1510, a partially hydrolyzedproduct) or soy protein isolate (not hydrolyzed).

The branched chain amino acids (BCAA) function to stimulate proteinsynthesis, reduce protein catabolism, activate muscle proteinrebuilding, reduce muscle fatigue, reduce muscle damage and theaccumulation of biomarkers associated with muscle cell damage or stress.In one embodiment, the branched chain amino acid is leucine. In certainembodiments, leucine, either as a separate amino acid or as part of aprotein, is present in an amount between about 2% and about 5% of thedietary supplement. In certain embodiments, the supplement contains atleast about 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%,3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%,4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8% or 4.9% total leucine. Incertain embodiments, the supplement contains up to about 2.1%, 2.2%,2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%,3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%,4.7%, 4.8%, 4.9% or 5% total leucine. In particular embodiments, thesupplement contains between about 2.5% and about 5% total leucine, orbetween about 3.5% and about 5% total leucine, or between about 3.5 andabout 4.5% or between about 3.6% and about 4.4% total leucine. Incertain embodiments, the supplement contains at least about 0.8%, 0.85%,0.9%, 0.95%, 1.0% 1.05%, 1.1%, 1.15%, 1.2% or 1.25% free leucine. Incertain embodiments, the supplement contains up to about 0.85%, 0.9%,0.95%, 1.0% 1.05%, 1.1%, 1.15%, 1.2% 1.25% or 1.3% free leucine. Inparticular embodiments, the supplement contains between about 0.8% and1.4% free leucine, or between about 0.9% and 1.3% free leucine.

In various embodiments, the composition further comprises one or moreadditional amino acids or their salts or derivatives, for example,glutamine, glutamic acid, one or more other BCAA (isoleucine, orvaline), or arginine. Each of these amino acids is considered to play arole in improving performance or influencing recovery from strenuousactivity, for example by shifting from protein catabolism to proteinbiosynthesis, by having a sparing effect on loss of an amino acid orprotein such as muscle protein, and/or by providing one or moreintermediates for energetic or biosynthetic purposes, such astricarboxylic acid (TCA) cycle intermediates.

In certain embodiments, the supplement comprises at least about 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36% or 37% fat. In certain embodiments, the supplement comprises upto about 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37% or 38% fat. In particular, the supplement cancomprise between about 24% and about 34% fat, or between about 26% andabout 32% fat, or between about 28% and about 30% fat.

The fat component of the dietary supplement typically comprises acombination of fats, at least one of which is a source of medium chaintriglycerides. In one embodiment, the source of medium chaintriglycerides is coconut oil, palm kernel oil, or a combination of thoseoils and other MCT-containing plant oils. In another embodiment,commercially available blends of medium chain triglycerides areutilized, e.g., NEOBEE® MCTs (Stepan Lipid Nutrition, Maywood, N.J.),among many others. In one embodiment, the composition comprises about15% to about 30% medium chain triglycerides as a percent of total fat.In one embodiment, the composition comprises about 2.6% to about 7.6%medium chain triglycerides as a percent of the total nutritional contentof the supplement. The remainder of the fat component can come from theprotein source, e.g., the meat component if present. Additionally oralternatively, fat sources, such as lard, poultry fat, vegetable oil,fish oil and the like, can be added.

The carbohydrate content should be kept low, and should comprise mostlycomplex carbohydrates such flour, meal or starch. In certainembodiments, the supplement comprises up to 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25%carbohydrate; however, some embodiments keep the carbohydrate contentlow, for instance, less than 20%, or less than 15%, or less than 10%, orless than 7%, of the total nutritional content of the supplement. Bykeeping the carbohydrate content comparatively low and/or by usingcomplex carbohydrates, insulin secretion can be minimized.

It should be kept in mind that the nutritional supplement is intended toprovide only a portion of the animal's total daily nutritional needs, inparticular, an amount of nutrients (i.e., combination of proteins,peptides, amino acids, fats, carbohydrates, micronutrients) sufficientto enhance exercise performance but not enough to cause the animal tofeel full or sluggish. Accordingly, the supplement can be formulated toprovide the animal with nutrients in amounts between about 1.0 g/kg bodyweight (BW) and about 3.0 g/kg BW of the animal. In certain embodiments,the supplement is formulated to provide the animal with nutrients inamounts of at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 or 2.9 g/kg BW of theanimal. In certain embodiments, the supplement is formulated to providethe animal with nutrients in amounts up to about 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9 or 3.0 g/kg BW of the animal. In one embodiment, the supplement isformulated to provide the animal with nutrients in amounts between about1.2 and about 2.2 g/kg BW of the animal. In another embodiment, thesupplement is formulated to provide the animal with nutrients in amountsbetween about 1.4 and about 2.0 g/kg BW of the animal. In a particularembodiment, the supplement is formulated to provide the animal withnutrients in amounts between about 1.6 and about 1.8 g/kg BW of theanimal.

The composition can be adapted for use in any form typical for dietarysupplements for animals. In one embodiment, the supplement is formulatedas pet food, such as in a pet treat form, such as a biscuit or chew. Inother embodiments, the nutrient content described above can be providedin the form of a gel, paste, jelly or beverage. Accordingly, themoisture or water content, or content of inert carriers can vary, aswould be appreciated by the skilled artisan.

The composition may further comprise one or more other agents to improveexercise performance, enhance metabolism, extend endurance and/orinfluence recovery from exercise. Performance enhancing agents and/orrecovery agents include antioxidants such as vitamin C, vitamin E; orvitamin A, compounds such as succinate or its salts or derivatives,various enzyme co-factors (e.g. coenzyme Q10), electrolytes such assodium, potassium, herbal supplements or extracts, and the like. In someembodiments, the compositions described herein may also be administeredor taken at about the same time as, or in conjunction with, suchadditional agents, or they can be formulated together in a singlecomposition, or in a single kit containing several compositions. Amongother things, such additional agents can also aid with the hydration orrehydration of the animal, as well as the oxygenation or reoxygenationof the animal's blood.

Another aspect of the invention features a method for improving exerciseperformance in an animal, particularly a canine. The method comprisesadministering to the animal a pre-exercise supplement comprising (a)about 35% to about 60% protein and/or amino acids, comprising one ormore structural proteins, one or more bioavailable proteins and one ormore branched chain amino acids; (b) about 20% to about 38% fat,comprising at least one source of medium chain triglycerides; and (c)about 5% to about 25% carbohydrate, in one aspect, comprising complexcarbohydrates.

In one embodiment, the supplement is administered prior to exercise, forinstance, between about zero and about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55 or 60 minutes prior to exercise. In one embodiment, thesupplement is administered about 10 minutes to about 20 minutes, orbetween about 20 minutes to about 40 minutes, or between about 40minutes to about 60 minutes prior to exercise. In a particularembodiment, the supplement is administered about 30 minutes prior toexercise. For extended exercise, the supplement can be administeredperiodically during exercise. For instance, additional supplement couldbe provided after 30, 60, 90 or more minutes of continuous exercise.

The method provides for administration of an effective amount of thecomposition for improving exercise performance. The effective amountrequired is an amount sufficient to accomplish one or more of thefollowing effects: (1) increased BCAA in the blood within about 30minutes following administration, (2) increased leucine in the bloodwithin about 30 minutes following administration; (3) increasedavailability of free fatty acids, and/or glycerol for exercisingmuscles, for instance, as measured by increase in blood circulatinglevels of those substances; (4) substantial avoidance of insulinsecretion following administration (e.g. less than 5%); (5) reduction ofactivity-induced protein catabolism; (6) increased protein biosynthesis(7) stable or increased blood oxygenation, (8) reduced production of atleast one stress hormone; (9) reduced production of protein oxidationproducts, (10) increased or reduced-depletion of endogenous pH bufferingagents like beta-alanine and/or carnosine levels to off-setexercise-induced lactic acid production, and/or (11) reduced fatigue orreduced soreness. Such levels can be measured by comparing levels withand without use of the present compositions before exercise as describedherein.

The method can be used in any animal or group of animals thatparticipates in physical activity, particularly those that are subjectedto strenuous activity such as working, training, competitive sports andthe like. In particular, the animals are canines, such as working dogs,competing dogs, or dogs that accompany their human caretakers on, forexample, walking, jogging, hiking, or running.

The methods of the invention for improving exercise performance byadministering the dietary supplement described herein can also involveadministration of additional agents for improving exercise performanceor another ancillary beneficial objective, such as enhancing recoveryfrom strenuous exercise or minimizing stress-related consequences ofexercise, as described above. The administration of the dietarysupplement for improving exercise performance can precede, or besimultaneous or sequential with, or can follow the administration of theother agents. For example, the pre-exercise supplement and one or morerecovery agents may be taken before the start of, during, or after thecompletion of the physical activity.

Another aspect of the invention features kits for improving exerciseperformance, and kits for preparing a dietary supplement as describedherein. The kits for improving exercise performance comprise, inseparate containers in a single package or in separate containers in avirtual package: (1) a pre-exercise supplement comprising (a) about 35%to about 60% protein and/or amino acids, comprising one or morestructural proteins, one or more bioavailable proteins and one or morebranched chain amino acids; (b) about 20% to about 38% fat, comprisingat least one source of medium chain triglycerides; and (c) about 5% toabout 25% carbohydrate, in one aspect, comprising complex carbohydrates;and (2) instructions for using the supplement for improving exerciseperformance in an animal that is about to undertake such activity.

The kits further or optionally comprise one or more recovery agents,additional compositions, or medicaments for improving exerciseperformance, and/or for influencing recovery from strenuous physicalactivity or for treating or mitigating damage resultant from strenuousphysical activity in animal. The kits also optionally or furthercomprise additional instructions for using the performing-enhancing orrecovery-aiding agents, additional compositions, or medicaments inconjunction with the pre-exercise supplement.

The kits further or optionally comprise instructions for one or more ofusing the kit for preparing the supplement or administering thesupplement to an animal. A vessel, bowl or container, or the like can beprovided in the kits for admixing the package contents to prepare thesupplement. For example a simple disposable, flexible mixing bag with areclosable or zip-type fastener may be very useful for admixing thecomponents. Alternatively, the kit may include instructions for admixingthe components as they are consumed such that the supplement is formedin the animal at the time of administration.

Still other aspects provided herein include means for communicatinginformation about, or instruction for use of, a dietary supplementuseful for improving exercise performance in an animal. The informationis communicated particularly about pre-exercise dietary supplementscomprising (a) about 35% to about 60% protein and/or amino acids,comprising one or more structural proteins, one or more bioavailableproteins and one or more branched chain amino acids; (b) about 20% toabout 38% fat, comprising at least one source of medium chaintriglycerides; and (c) about 5% to about 25% carbohydrate. Theinformation communicated by the described means is about, or theinstructions are for, one or more of:

-   -   (1) instructions for administering the supplement to an animal        before exercise,    -   (2) information on providing proper nutrition, including the        supplement, to an animal that exercises or that will be        exercising,    -   (3) information about exercise, including physical activity or        strenuous physical activity,    -   (4) information regarding physical, cellular or biochemical        results of exercise, or nutrients required during exercise, or        the uptake of nutrients prior to exercise, or    -   (5) comparative information or test results regarding the        supplement.

The communication means typically comprises a physical or electronicdocument, digital storage media, optical storage media, audiopresentation, audiovisual display, or visual display containing theinformation or instructions. In various embodiments, the communicationmeans is selected from the group consisting of a displayed web site,visual display kiosk, brochure, product label, package insert,advertisement, handout, public announcement, audiotape, videotape, DVD,CD-ROM, computer readable chip, computer readable card, computerreadable disk, USB device, FireWire device, computer memory, and anycombination thereof.

Also provided herein is a package comprising a composition of theinvention and a label, logo, graphic, symbol, slogan, or the likeidentifying the package and composition within as useful for improvingexercise performance in an animal. In one embodiment, the packagecomprises a pre-exercise dietary supplement having (a) about 35% toabout 60% protein and/or amino acids, comprising one or more structuralproteins, one or more bioavailable proteins and one or more branchedchain amino acids; (b) about 20% to about 38% fat, comprising at leastone source of medium chain triglycerides; and (c) about 5% to about 25%carbohydrate. The package further contains a word or words, picture,design, logo, graphic, symbol, acronym, slogan, phrase, or other device,or combination thereof, either directly on the package or on a labelaffixed thereto, indicating that the contents of the package are usefulfor improving exercise performance in an animal. In some embodiments,the package can comprise the words “improves exercise performance”,“extends endurance”, “stimulates metabolism”, or an equivalentexpression printed on the package. Any package or packaging materialsuitable for containing the composition is useful in the invention,e.g., a bag, box, bottle, can, pouch, and the like manufactured frompaper, plastic, foil, metal, and the like.

In another aspect, the invention provides for use of one or morecomposition provided hereinabove to prepare a medicament for improvingexercise performance in an animal, particularly a canine. The medicamentcan further comprise one or more performance enhancing or exerciserecovery agents, vitamins, electrolytes, antioxidants, herbal extracts,NSAIDs, analgesics or pain medication, or combinations thereof.Generally, medicaments are prepared by admixing a compound orcomposition with excipients, buffers, binders, plasticizers, colorants,diluents, compressing agents, lubricants, flavorants, moistening agents,and other ingredients known to skilled artisans to be useful forproducing medicaments and formulating medicaments that are suitable foradministration to an animal.

The invention can be further illustrated by the following example,although it will be understood that this example is included merely forpurposes of illustration and is not intended to limit the scope of theinvention unless otherwise specifically indicated.

Example 1

Feeding studies with a pre-exercise food were conducted to establishefficacy and to determine the feeding dose and timing recommendationprior to exercise. The objectives of the studies were: (1) to determinean effective feeding dose of a pre-exercise snack by assessing theappearance of key nutrients and metabolites of dietary constituents inthe blood of dogs after feeding; and (2) to determine an effectivefeeding time prior to exercise using the feeding dose selected inObjective 1.

Methodology

A feeding trial was performed using adult dogs to assess thebioavailability of the amino acid leucine and total branched-chain aminoacids after ingesting the test food.

A timing trial with no exercise was conducted—all dogs (body weight (BW)mean: 22 kg+/−2.7 kg) were fasted overnight, but had free access towater. Three treatment groups were utilized, each consisting of 15 dogs.Two test groups were fed 1.6 g of food/kg BW of either Chicken and ricedry extruded kibble formulated to contain at least 30% crude protein andat least 20% crude fat representative of a canine performance food(Nestle Purina, St. Louis Mo.) or test food at approximately 9 am. Athird control group was included to represent not feeding anything priorto exercise (fasted). Blood samples were collected before feeding and atvarious times after feeding (0, 30, and 60-min post ingestion).

Test Diets:

-   -   Control Food—Chicken and rice dry extruded kibble formulated to        contain at least 30% crude protein and at least 20% crude fat        representative of a canine performance food. Targeted to contain        31% crude protein on as fed basis.    -   Test Food—formulated to contain 44% crude protein on as-fed        basis.

Table 1 below summarizes the formula ingredients and percentages of thetest formula used in the feeding trial.

TABLE 1 test food ingredients Test formula Beef Hearts 60.5 L-leucine0.9 Soy Grits 80-0 5.0 Coconut Oil 7.2 Soy Protein Modified (SPI 1510)5.5 Whey Protein Concentrate 6.0 Glycerine 5.0 GDL NE 1.2 Flavor SmokeP-50 0.3 Calcium Propionate 0.12 Salt 1.5 Phosphoric Acid 0.5 CaramelColoring 0.75 Garlic Powder 0.5 Soy Protein Isolate 2.0 TG Gelatin 1.5Soy Lecithin 2.0 Sorbic Acid 0.28 PMX Naturox Plus 0.1 Vitamin mix 0.125

Trial Results

Test Foods and Protein Ingestion:

The mean food ingestion amounts are listed below for each treatmentgroup.

As fed basis Dry matter basis L-Leu ingested basis Control food −1.6g/kg BW 1.5 g/kg BW 56.8 mg/kg BW Test formula  1.6 g/kg BW 1.2 g/kg BW56.6 mg/kg BW

The mean crude protein (on dry matter basis) ingestion amounts arelisted below for each treatment group.

Dry matter basis Control food −0.49 g/kg BW Test food  0.53 g/kg BW

Effect of Test Food on L-Leucine Bioavailability:

The trial evaluated the postprandial appearance of amino acids in dogserum after ingesting 1.6 g of food/kg BW (Test Food or Control Food) orwithout food. Results are shown in Table 2 below.

TABLE 2 Mean (+/− SEM) serum L-leucine concentration for treatmentgroups. Treatment Groups Time after Control—No Control— Test— feedingfeeding group feeding group feeding group ANOVA P- (minutes) FastedControl Food Prototype Food value serum leucine concentration (nmol/mL)0 178.1 +/− 7.4 172.6 +/− 9.6 188.5 +/− 8.3 NS¹ 30 163.9 +/− 7.4 170.9+/− 7.5 191.7 +/− 7.2 <0.05 60 166.3 +/− 7.3  201.7 +/− 11.3 197.5 +/−8.0 <0.05 serum branched-chain amino acid concentration (nmol/mL) 0 479+/− 15 480 +/− 26 499 +/− 16 NS¹ 30 452 +/− 18 479 +/− 22 499 +/− 11 NS¹60 461 +/− 17 528 +/− 27 507 +/− 18 NS¹ ¹NS: not statisticallysignificant

The data specifically demonstrate that the test food formula is superiorin its ability to significantly enhance the delivery of L-Leu (ANOVAP<0.05), as indicated by the 12.2% increase serum appearance of theamino acid at 30 min after ingestion compared to a similar amount of amain-meal performance formula (Control Food). Both test foods fed at 1.6g of food per kg BW actually deliver the same amount of dietaryL-leucine to the animal, but the test formula is uniquely capable ofenhancing L-leucine bioavailability to the animal at 30 min afteringestion.

In addition, the data specifically demonstrate that the test foodformula is superior in its ability to quantitatively enhance thedelivery of total branched-chain amino acids, as indicated by the 4%increase serum appearance of the amino acid at 30 min after ingestioncompared to a similar amount of a main-meal performance formula (ControlFood).

Summarized Benefits from Trials

The studies above can be summarized as follows:

-   -   Effective dose is approximately 1.6 g to 1.8 g of test food per        kg of body weight. This equates to approximately a 36-40 gram        bar for a 50 lb dog.    -   Effective feeding time is approximately 30 minutes prior to        exercise.

Example 2

A feeding trial was conducted using an experimental design and controland test formulas similar to those described in Example 1, except dogswere exercised in this trial.

The trial evaluated the postprandial appearance of amino acids in dogserum after ingesting 1.6 g of food/kg BW (Test Food Formula or ControlFood) or without food. Feeding of the test or control food occurred 60minutes before exercise. Exercise at a 5-6 mph pace was maintained for90 minutes. The data below were obtained from blood collected 30 minbefore exercise, immediate before exercise, and 60 minutes after startof exercise.

The data specifically demonstrate that serum leucine concentrations aresignificantly lower in dogs that do not receive a pre-exercise food.Either the test food or control food formula results in preventing theexercise-induced decrease in blood leucine concentration after 60 min ofexercise. Both the control food and the test food were fed at 1.6 g offood per kg BW. However, the test food only contained 2.72% leucine,whereas the control food contained 3.57% leucine. Even so, the feedingresulted in similar levels of blood leucine before and during exercise.(Table 3).

-   -   Protein content of test formula: 36.4% on As fed basis, 44.66%        on dry matter basis    -   Protein content of control formula: 30.7% on As fed basis,        33.04% on dry matter basis

TABLE 3 Mean (+/− SEM) serum L-leucine and BCAA concentrations fortreatment groups Treatment Groups Time relative Control—No Control—Test— ANOVA to exercise feeding group feeding group feeding group P-(minutes) Fasted Control Food Prototype Food value serum leucineconcentration (nmol/mL) −30 154.7 +/− 8.0 159.2 +/− 9.3 161.6 +/− 5.5NS¹ 0 152.5 +/− 7.7 167.0 +/− 6.3 167.0 +/− 5.6 NS¹ 60 131.6 +/− 6.2156.4 +/− 7.4 150.8 +/− 5.6 <0.05 serum branched-chain amino acidconcentration (nmol/mL) 0 466 +/− 16 479 +/− 20 484 +/− 14 NS¹ 30 465+/− 20 502 +/− 16 504 +/− 16 NS¹ 60 426 +/− 18 472 +/− 18 479 +/− 170.08 ¹NS: not statistically significant

Example 2

An exercise trial was performed using adult dogs to assess the ingestionof a protein-fat-rich nutritional supplement prior to exercise inaccordance with one embodiment of the present disclosure. Specifically,data contained in this document was based on a formula containingapproximately 4.3% total L-leucine (as fed basis; 5.5% on DM basis)compared to 3.6% total L-leucine (as fed basis; 4.6% on DM basis)contained in previously examined formulas. Consequently, the ingesteddose amount of L-leucine increases from 57 mg/kg BW to 68 mg/kg BW. Bycontrast, total protein content was 1.5% lower on DM basis compared topreviously examined formula (data summarized below).

The present example generates data related to delivery of key nutrientsbefore and during exercise when feeding a pre-exercise food beforeexercise and generates additional exercise related data to supportmetabolic and physiological benefits related to improving exerciseperformance and/or metabolism during exercise.

Summarized Benefits from Trials

The studies above can be summarized as follows:

-   -   composition reduces protein breakdown and amino acid oxidation,    -   composition reduces muscle cell disruption and cell stress,    -   composition reduces liver cell disruption and cell stress,    -   composition reduces protein oxidation by reactive nitrogen        species,    -   composition to improve muscle cell buffering capacity and/or        serum carnosine concentrations,    -   composition to improve beta-alanine concentrations to help        promote endogenous carnosine synthesis, and    -   composition to improve serum osmolyte concentrations and/or        serum taurine concentrations.

Methodology Animals and Treatments:

Exercise trial was conducted using Husky x Pointer cross-bred dogs(N=38; 2 to 9 yrs old; mean 4.7 yrs+1-2.2 SD; BW mean: 23.7 kg+1-3.3SD). Dogs were selected to participate in the trial and allocated evenlyinto 3 treatment groups to account for age, BW, and exercise ability.

All dogs were exercise-conditioned for 3 weeks prior to the treatmentphase. Conditioning consisted of using an exercise wheel at 7-8 mph 2 or3 times a week, for 3 weeks. Duration of exercise was graduallyincreased over the 3 week conditioning period to ensure all dogs couldrun for the 2-hr duration of the exercise during the treatment phase.

Experimental treatments consisted of a control group (N=13) that did notany receive pre-exercise food and 2 test-food groups that received a drykibble food similar to a main meal chicken and rice formula (1.6 g perkg BW; N=13) or a test formula (ProPlan Prime bar; 1.6 g per kg BW;N=13) at 30 minutes prior to the start of exercise. All dogs wereregularly fed once-daily and had free access to water.

Ambient environmental temperature during exercise ranged from 50 to 65°F.

Exercise Treatment Phase:

The treatment phase included a 2-hr exercise bout on days 1, 4, 7, 10,13, 17, 18, and 19. Effects of a single day of exercise (1-d) wereevaluated by collecting blood samples on day 7. Effects of 3 consecutivedays of exercise were evaluated by collecting blood samples on day 19(3-d). With one exception, all exercise bouts were performed using theexercise wheel for 2 hours at 7-8 mph. The one exception was on day 18during the 3-day exercise test, in which dogs were exercised for 25 minat 13 mph while running as a group in harness. All dogs were dividedinto 4 exercise groups of 9 or 10 dogs per group, in which all 3treatments were represented in each exercise group. On blood samplingdays (day 7 and 19), dogs were temporarily stopped after the initial 60min of exercise to obtain a blood sample and then run in the reversedirection for remainder of the exercise.

Four blood samples were collected on day 7 and day 19; 1.) immediatelybefore ingesting the pre-exercise treatment food (30 min prior toexercise), 2.) 30 min after ingesting pre-exercise food (immediatelybefore onset of exercise), 3.) after 60 min of exercise, and 4.) after120 min of exercise (end of exercise).

Test Diets:

The diets are outlined as follows:

-   -   Control Food—representative of a commercial main meal food        containing Chicken and Rice. Targeted to contain:        -   31-33.5% crude protein on as fed basis (33.7% on DM basis)        -   28-35% carbohydrates on as fed basis (34.4% on DM basis)        -   20-23% crude fat on as fed basis (23.3% on DM basis)    -   Test Food—formulated to contain:        -   32% crude protein on as fed basis (42% on DM basis)        -   23-25% crude fat on as fed basis (30-32% on DM basis)

Test Foods and Protein Ingestion:

The mean food ingestion amounts are listed below for each treatmentgroup.

As fed basis Dry matter basis Control food 1.6 g/kg BW 1.5 g/kg BW Testformula 1.6 g/kg BW 1.2 g/kg BW

The mean crude protein ingestion amount per body weight listed below foreach treatment group.

Control food −0.49 g/kg BW Test formula −0.51 g/kg BW

The mean L-leucine (on as fed and dry matter basis) ingestion amountsare listed below for each treatment group.

Control food −56.8 mg/kg BW Test formula −68.0 mg/kg BW

Trial Results

The trial evaluated the appearance of 3-methylhistidine (3MH) andcreatine kinase in dog serum as biomarkers of muscle protein breakdownand exercise-induced muscle cell disruption, respectively. Theappearance of asparagine aminotransferase (AST) is generally a biomarkeroriginating from the muscle and liver, and related to cellulardisruption and damage (Banff et al., 2012). Exercise naturally resultsin a greater oxidative load, which in-part, causes cell membranedisruption and leakage (cell stress), which is demonstrated by observingincreases in various muscle-specific enzymes, metabolites, and/orelectrolytes in blood; some of which have become hallmark indicators ofexercise-induced muscle-fiber damage and disruption of membraneintegrity, such as lactate dehydrogenase (LDH) and creatine kinase (CK;Banfi et al., 2012). Dogs performing prolonged endurance-relatedexercise (Strasser et al., 1997; Davenport et al., 2001; Wakshlag et al.2004; McKenzie et al., 2007), short duration (less than 2 min) sprinting(Lassen et al., 1986; Snow et al., 1988; Rose et al., 1989; Ilkiw etal., 1989; Rovira et al., 2007), or repetitive retrieving (durationapproximately 10 min; Matwichuk et al., 1999; Steiss et al., 2004;Steiss et al., 2008) experience exercise related changes in varioushematological analytes. These exercise-induced changes are largelyreported as being within clinically “normal” ranges, yet many arestatistically different from resting levels and may be associated withincreased oxidative stress. Consequently, monitoring these blood markersprovides a basis for assessing how dietary interventions may offsetnatural metabolic and physiological stresses associated with exercise.

Protein Catabolism: The data demonstrates that the test food formulacompared to a control formula is superior in its ability to reduceprotein catabolism, as indicated by maintaining lower serumconcentrations of 3MH before the start of exercise by 10% and by 7-10%during a single day of exercise (Table 4). The test food formula alsoreduces protein catabolism by 5% compared to fasted dogs before theonset of exercise. Feeding the control food as a pre-exercise supplementappears to increase protein catabolism during exercise, as compared todogs fed the test food and fasted control dogs.

In addition, after continued exercise conditioning with or withoutpre-exercise supplementation for 3 weeks, which was followed by a 3-dexercise challenge, 3MH serum concentrations on d-3 in dogs fed the testfood were 9% lower before exercise and maintained 12% lower after 60 minof exercise compared to dogs fed the control food. The test food formulaprovides an even greater reduction of protein catabolism compared tofasted dogs, as 3MH was about 15% lower before the onset of exercise andremained at least 7% lower during exercise

Muscle Cell Disruption/Muscle Damage Marker: The test food formulacompared to a control formula is superior in its ability to supportreduced muscle damage during exercise, as indicated by a lower serumconcentrations of CK before the start of the 1-d exercise (26%) and by29% during a single 2-hour bout of exercise (Table 4). Feeding thecontrol food as a pre-exercise supplement appears to result in CKconcentrations to be slightly elevated before starting exercise, and ishigher than fasted dogs during and after exercise, as compared to fastedcontrol dogs.

After continued exercise conditioning for 3 weeks with or withoutpre-exercise supplementation, which was followed by a 3-d exercisechallenge, CK concentrations in serum on d-3 were relatively similarbefore exercise, but after 2 hrs of exercise ingestion of eithertreatment food resulted in a reduced CK concentrations by about 40%compared to dogs fasted prior to exercise.

AST serum concentrations were 8-15% lower at all sample time pointsbefore and during a single day exercise in dogs fed the test foodformula compared to either the fasted dogs or the dogs fed the controlfood. On d-3 of the 3-d exercise challenge, AST concentrations followeda similar response to CK concentrations, in which dog fed eithertreatment group had 32% lower AST concentrations at the end of the 2-hrexercise.

Amino Acid Oxidation and Ureagenesis:

Amino acids derived from the digestion of dietary protein contribute toa highly active intracellular pool of amino acids, which that cannot beexpanded. Excess amino acids from dietary protein and/or endogenousprotein breakdown increase this amino acid pool. Consequently the excessamino acids follow 3 different paths; 1. new protein synthesis, 2.oxidation for energy that yields urea synthesis for disposal of nitrogen(ureagensis), and/or 3. Conversion to other compounds (reviewed bySchutz, 2011). A relationship exists in which the higher the dietaryprotein, thus excess amino acids being oxidized, the higher the ureaproduction.

Serum urea concentrations on the 1-d exercise challenge were similarbefore exercise, but became elevated by 60-min of exercise in the dogsfed either of the pre-exercise supplement bars compared to the fastedcontrol dogs. Dogs fed the control food had 10% higher ureaconcentration at the end of the 2-hr exercise compared to fasted controldogs, whereas dogs fed the test food were elevated by only 6%. Thisindicates that the protein in the control food was more oxidized duringexercise, which is consistent with the observed elevation in 3 mH in thedogs fed the control food, thus less impactful to reduce proteincatabolism compared to the test food.

After continued exercise conditioning for 3 weeks with or withoutpre-exercise supplementation, which was followed by a 3-d exercisechallenge, mean urea concentrations in serum on d-3 was highest in thecontrol food group before exercise and during exercise. Specifically,dogs fed the control food had 5% higher serum urea compared to eithertest food group or fasted control. By 60 min of exercise, ureaconcentration in the dogs fed the control food was 8% and 11% highercompared to dogs fed the test food or fasted. Thus dogs fed the controlfood before exercise are metabolizing amino acids at a higher level, butbecause 3-mH levels are also elevated, indicates that ingestion of theprotein composition in the control food does not reduce proteincatabolism during exercise.

TABLE 4 Mean (+/−SD) canine serum 3-methyl histidine, creatine kinase,Asparagine aminotransferase, and urea concentration at different timesrelative to exercise for each treatment groups Treatment Groups Timerelative to Control− Control− Test− exercise (minutes) fasted group foodgroup food group serum 3-methyl histidine-(ug/mL) 1-day exercise bout−30 1.61 +/− 0.50 1.61 +/− 0.45 1.66 +/− 0.55 0 1.58 +/− 0.52 1.67 +/−0.89 1.50 +/− 0.52 60 1.67 +/− 0.55 1.80 +/− 0.45 1.62 +/− 0.41 120 1.68+/− 0.46 1.87 +/− 0.44 1.73 +/− 0.50 day 3 of 3-day exercise bout −301.84 +/− 0.53 1.73 +/− 0.55 1.57 +/− 0.42 0 1.80 +/− 0.52 1.66 +/− 0.501.51 +/− 0.38 60 1.79 +/− 0.50 1.78 +/− 0.54 1.57 +/− 0.39 120 1.86 +/−0.55 1.75 +/− 0.53 1.72 +/− 0.40 serum creatine kinase concentration(U/L) 1-day exercise bout −30 63 +/− 19 84 +/− 31 62 +/− 13 0 65 +/− 2275 +/− 29 63 +/− 17 60 98 +/− 38 118 +/− 42  94 +/− 30 120 112 +/− 33 147 +/− 75  104 +/− 36  day 3 of 3-day exercise bout −30 74 +/− 21 81+/− 25 76 +/− 10 0 80 +/− 27 85 +/− 31 81 +/− 13 60 111 +/− 39  115 +/−37  107 +/− 15  serum asparagine aminotransferase concentration (U/L)1-day exercise bout −30 21 +/− 9 21 +/− 12 18 +/− 9 0 22 +/− 9 22 +/− 1119 +/− 9 60  31 +/− 10 30 +/− 11  28 +/− 10 120 35 +/− 9 35 +/− 12  29+/− 10 day 3 of 3-day exercise bout −30 23 +/− 7 23 +/− 4 22 +/− 5 0 23+/− 7 23 +/− 6 23 +/− 6 60  32 +/− 11 29 +/− 6 29 +/− 7 120  34 +/− 1223 +/− 5 23 +/− 6 serum urea concentration-(mg/dL) 1-day exercise bout−30 508 +/− 86 509 +/− 166 519 +/− 137 0 496 +/− 75 493 +/− 156 502 +/−128 60  528 +/− 104 566 +/− 81  546 +/− 126 120  553 +/− 105 608 +/− 103587 +/− 156 day 3 of 3-day exercise bout −30 478 +/− 65 500 +/− 181 505+/− 132 0 495 +/− 61 508 +/− 123 518 +/− 124 60 474 +/− 60 529 +/− 113513 +/− 137 120 479 +/− 73 547 +/− 132 551 +/− 166

Liver Cell Disruption: The trial evaluated the appearance of alanineaminotransferase (ALT), which is largely a biomarker originating fromthe liver, but also in smaller amounts in the kidneys, heart, muscles,and related to cellular disruption and damage (Banff et al., 2012). Thedata demonstrates that the test food formula compared to a controlformula or fasted control dogs is superior in its ability to reduceliver cell disruption, as indicated by maintaining lower serumconcentrations of ALT before the start of a single bout of exercise byat least 7 and 9%, respectively. After 60 and 120 min of exercise, ALTconcentrations remained low in dogs fed the test food, as dogs fed thecontrol food or fasted had 11 and 11% higher concentrations,respectively (Table 5). With continued exercise conditioning with orwithout pre-exercise supplementation for 3 weeks, which was followed bya 3-d exercise challenge, pre-exercise serum ALT concentrations wereelevated in all groups. However, dogs fed the test food had slightlylower ALT (3-5%) concentration before the start of exercise. Dogs fedthe test food were 3.5 and 4.5% lower after 60 and 120 min of exercise,respectively, compared to dogs fed the control food. The test foodformula provides an even greater difference compared to fasted dogs, asALT was 10 and 12% lower after 60 and 120 min of exercise, respectively.

TABLE 5 Mean (+/− SD) canine serum alanine aminotransferaseconcentration at different times relative to exercise for each treatmentgroups. serum alanine aminotransferase Control— Control— Test—concentration (U/L) fasted group food group food group 1-day exercisebout −30 48 +/− 34 48 +/− 19 44 +/− 16 0 47 +/− 34 53 +/− 33 44 +/− 1660 52 +/− 36 52 +/− 21 46 +/− 16 120 52 +/− 37 52 +/− 21 46 +/− 16 day 3of 3-day exercise bout −30 61 +/− 22 59 +/− 19 58 +/− 25 0 60 +/− 22 59+/− 21 57 +/− 24 60 65 +/− 25 61 +/− 22 58 +/− 24 120 65 +/− 25 59 +/−20 57 +/− 24

Protein Oxidation by Reactive Nitrogen Species: Nitric oxide (NO) inexcess reacts with superoxide radical and produces stable and highlyreactive peroxynitrite. The latter causes the nitration of tyrosineresidues of proteins to form 3-nitrotyrosine (3-NT) and thereby altersthe biological functions of proteins (Crow and Beckman, 1995, 1996).Formation of nitrotyrosine is often thought to be accompanied with acuteor chronic inflammation disease, whereby level of nitric oxide iselevated (reviewed by Cai and Yan, 2013). Moreover, 3-NT gains itsimportance in pathological manifestations as it causes oxidative lesionsto DNA bases (Murata and Kawanishi, 2004). Other than disease conditionsand inflammation, the exhaustive exercise also causes cellular damagedue to increased 3-NT production as a consequence of nitrosative stress.Earlier a study on human subjects performing exercise of unaccustomedintensity has documented an elevated serum and urinary 3-NT level andsuggested that 3-NT might act as an important diagnostic tool toexercise-induced damage of varied intensity (Radak et al., 2003). Recentexercise research in people further demonstrated that increasedformation of 3NT significantly correlated with increasing DNA damage(Sinha et al., 2010).

The trial evaluated the appearance of 3-nitrotyrosine (3NT) in serumbefore and during exercise. The data demonstrates that the test foodformula compared to a control formula or fasted control dogs is superiorin its ability to minimize 3-NT formation, as indicated by maintaininglower serum concentrations of 3-NT before the start of exercise by about24-36% and by 37% at the end of a single 2-hr bout of exercise comparedto dogs fed the control food (Table 6). Dogs fed the test food had lower3NT concentrations compared to fasted control dogs, and demonstrated thelowest 3NT concentration at the end of exercise. Dogs fed the controlfood had the highest concentration of 3NT even compared to fastedcontrol dogs.

With continued exercise conditioning with or without pre-exercisesupplementation for 3 weeks, which was followed by a 3-d exercisechallenge, pre-exercise 3NT concentrations on d-3 was 50-60% lower indogs fed the test food. After 60-min of exercise, 3NT in fasted controldogs and dogs fed the control food was 39 and 37% higher compared todogs fed the test food. At the end of exercise, 3NT concentrations werestill 29-35% higher compared to dogs fed the test food.

TABLE 6 Mean (+/− SD) canine serum 3-nitrotyrosine concentration atdifferent times relative to exercise for each treatment groups. serum3-nitrotyrosine Control— Control— Test— (ug/mL) fasted group food groupfood group 1-day exercise bout −30 1.7 +/− 0.9 2.0 +/− 1.3 1.5 +/− 0.8 01.7 +/− 0.8 2.4 +/− 1.6 1.5 +/− 0.7 60 2.1 +/− 1.4 2.7 +/− 1.9 1.9 +/−1.2 120 2.5 +/− 2.0 2.8 +/− 1.9 1.7 +/− 0.7 day 3 of 3-day exercise bout−30 1.2 +/− 1.1 1.6 +/− 2.0 0.6 +/− 0.2 0 1.3 +/− 1.0 1.4 +/− 1.3 0.9+/− 0.4 60 3.7 +/− 2.2 3.6 +/− 1.5 2.2 +/− 0.9 120 2.8 +/− 1.8 3.0 +/−1.5 2.0 +/− 0.8

Cellular pH buffering metabolites: Carnosine is an endogenouslysynthesized dipeptide of beta-alanine and histidine and is found in highconcentrations in skeletal muscle. Because carnosine is inert tointracellular enzymatic hydrolysis and does not contribute toproteogenesis, it functions as a stable intracellular buffer,particularly at the pH range of high intensity exercise. Carnosinesynthesis is rate-limited on the dietary availability of beta-alanine.In humans, increased levels of carnosine, through b-alaninesupplementation, have been shown to increase exercise capacity andperformance of several types of high-intensity exercise (Harris andStellingwerff, 2013). Another beta-amino acid with benefits for exerciseis taurine, which is demonstrated to help regulate cell hydration (Lang,2011) and provide other physiological functions that provideanti-inflammatory and anti-oxidant support (Ripps and Shen, 2012; Ra etal., 2013)

The trial evaluated the appearance of carnosine and beta-alanine inserum before and during exercise. The data demonstrates that the testfood formula compared to a control formula or fasted control dogs issuperior in its ability to maintain higher serum concentrations ofcarnosine and b-alanine 30 min after ingestion. During exercise,carnosine concentrations were similar for dogs fed either the test foodor control food, and these concentrations were 8.7 and 7% highercompared to the fasted control dogs after 60 and 120 min of exercise,respectively (Table 7). Dogs fed the test food had the highest serumb-alanine concentration before and during exercise compared to both thefasted control dogs and dogs fed the control food.

With continued exercise conditioning with or without pre-exercisesupplementation for 3 weeks, which was followed by a 3-d exercisechallenge, mean pre-exercise carnosine concentration on d-3 was at least9% higher in dogs fed the test food. After 60-min of exercise, carnosineconcentration was the highest in dogs fed the test food by 7 and 12%compared to dogs fed control food and fasted, respectively. At 120-minpost exercise, dogs fed either of the pre-exercise foods had higherserum carnosine concentration compared to fasted control dogs.

Dogs fed either of the pre-exercise foods had higher serum b-alanineconcentrations compared to fasted control dogs, as well as duringexercise.

The trial evaluated taurine concentration in serum before and duringexercise. The data demonstrates that the test food formula compared to acontrol formula or fasted control dogs is superior in its ability topromote at least 6% higher serum concentrations of taurine 30 min afteringestion at the start of a single 2-hr bout of exercise.

With continued exercise conditioning with or without pre-exercisesupplementation for 3 weeks, which was followed by a 3-d exercisechallenge, mean pre-feeding and pre-exercise taurine concentration ond-3 was at least at least 6% higher in dogs fed the test food comparedto dogs fed the control food and 14% higher compared to fasted control.At 30 min postprandial and immediately before starting exercise, taurineconcentration was at least 7% higher in dogs fed the test food. After60-min of exercise, taurine concentration was the highest in dogs fedthe test food by 17 and 13% compared to dogs fed control food andfasted, respectively.

TABLE 7 Mean (+/− SD) canine serum carnosine, b-alanine, and taurineconcentration at different times relative to exercise for each treatmentgroups. serum carnosine Control— Control— Test— (ug/mL) fasted groupfood group food group 1-day exercise bout −30 6.99 +/− 1.72 7.29 +/−1.43 7.34 +/− 0.65 0 6.57 +/− 1.60 6.47 +/− 2.02 7.04 +/− 1.06 60 8.16+/− 2.18 8.89 +/− 1.18 8.86 +/− 1.72 120 9.10 +/− 2.30 9.63 +/− 1.579.74 +/− 1.57 day 3 of 3-day exercise bout −30 6.40 +/− 1.41 6.81 +/−1.62 7.15 +/− 0.77 0 6.31 +/− 1.31 6.54 +/− 0.82 6.80 +/− 0.55 60 7.27+/− 1.88 7.61 +/− 1.40 8.12 +/− 1.15 120 7.60 +/− 1.79 8.75 +/− 1.258.76 +/− 1.18 Serum beta-alanine (ug/mL) 1-day exercise bout −30 0.54+/− 0.14 0.54 +/− 0.16 0.57 +/− 0.12 0 0.46 +/− 0.12 0.49 +/− 0.17 0.53+/− 0.12 60 0.59 +/− 0.24 0.64 +/− 0.15 0.68 +/− 0.16 120 0.62 +/− 0.170.62 +/− 0.18 0.65 +/− 0.12 day 3 of 3-day exercise bout −30 0.79 +/−0.17 0.92 +/− 0.20 0.90 +/− 0.10 0 0.59 +/− 0.16 0.64 +/− 0.26 0.63 +/−0.21 60 0.88 +/− 0.18 0.98 +/− 0.19 0.97 +/− 0.18 120 0.95 +/− 0.22 1.05+/− 0.24 0.98 +/− 0.21 Serum taurine (ug/mL) 1-day exercise bout −3023.8 +/− 4.8 25.3 +/− 8.6 26.1 +/− 5.8 0 24.1 +/− 4.9  25.9 +/− 11.727.5 +/− 7.2 60 19.3 +/− 6.1 20.8 +/− 4.7 20.1 +/− 8.5 120 17.7 +/− 3.719.6 +/− 7.9 18.7 +/− 7.1 day 3 of 3-day exercise bout −30 17.5 +/− 3.818.8 +/− 7.2 20.0 +/− 3.8 0 17.1 +/− 3.0 18.4 +/− 7.1 19.7 +/− 3.4 6016.0 +/− 3.5 15.4 +/− 4.6 18.1 +/− 5.1 120 15.2 +/− 7.3 13.6 +/− 3.714.3 +/− 2.3

The specification has disclosed typical embodiments of the invention.Although specific terms are employed, they are used in a generic anddescriptive sense only, and not for purposes of limitation. The scope ofthe invention is set forth in the appended claims. The skilled artisanwill appreciate that many modifications and variations of the claimedinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims theinvention may be practiced otherwise than as specifically described.

What is claimed is:
 1. A canine pre-exercise supplement comprising: a.about 35% to about 60% protein or amino acids, comprising one or morestructural proteins, one or more bioavailable proteins and one or morebranched chain amino acids; b. about 20% to about 38% fat, comprising atleast one source of medium chain triglycerides; and c. about 5% to about25% carbohydrate.
 2. The supplement of claim 1, formulated to providethe canine with (a) between about 1.2 g/kg BW and about 2.0 g/kg BWtotal nutrition.
 3. The supplement of claim 1, wherein the structuralprotein includes animal muscle.
 4. The supplement of claim 3, whereinthe muscle is heart muscle.
 5. The supplement of claim 1, wherein thebioavailable protein is selected from whey, partially hydrolyzed soy andhydrolyzed amino acids, or any combination thereof.
 6. The supplement ofclaim 1, wherein the branched chain amino acids include L-leucine. 7.The supplement of claim 1, wherein the source of medium chaintriglycerides is coconut oil.
 8. The supplement of claim 1, wherein thecarbohydrate comprises complex carbohydrates.
 9. The supplement of claim1, comprising beef heart, soy meal and soy protein.
 10. The supplementof claim 9, comprising coconut oil and L-leucine.
 11. A method forimproving exercise performance in a canine, comprising: a. identifying acanine that will be performing exercise; and b. administering to thecanine a pre-exercise supplement in an effective amount, thepre-exercise supplement comprising (i) about 35% to about 60% protein oramino acids, comprising one or more structural proteins, one or morebioavailable proteins and one or more branched chain amino acids; (ii)about 20% to about 38% fat, comprising at least one source of mediumchain triglycerides; and (iii) about 5% to about 25% carbohydrate,wherein the supplement is formulated to provide the canine or withbetween about 1.2 g/kg BW and about 2.0 g/kg BW total nutrition, whereinthe supplement is administered to the canine between about zero andabout 60 minutes prior to exercise.
 12. The method of claim 11, whereinthe supplement is administered to the canine between about zero andabout 30 minutes prior to exercise.
 13. The method of claim 11, whereinthe structural protein in the supplement includes animal muscle.
 14. Themethod of claim 13, wherein the muscle is heart muscle.
 15. The methodof claim 11, wherein the bioavailable protein in the supplement isselected from whey, partially hydrolyzed soy and hydrolyzed amino acids,or any combination thereof.
 16. The method of claim 11, wherein thebranched chain amino acids in the supplement include L-leucine.
 17. Themethod of claim 11, wherein the source of medium chain triglycerides inthe supplement is coconut oil.
 18. The method of claim 11, wherein thecarbohydrate in the supplement comprises complex carbohydrates.
 19. Themethod of claim 11, wherein administration of the supplement does notcause a substantial increase in blood insulin.
 20. The method of claim11, wherein, relative to a control animal not receiving the supplement,the blood level of branched chain amino acids is increased 30 to 60minutes after the animal ingests the supplement.
 21. The method of claim11, wherein the supplement comprises leucine and, relative to a controlanimal not receiving the supplement, the blood level of the leucine isincreased 30 to 60 minutes after the animal ingests the supplement. 22.The method of claim 11, wherein the effective amount provides at leastone of the following: (1) increased BCAA in the blood within 30 minutesfollowing administration, (2) increased leucine in the blood within 30minutes following administration; (3) increased availability of freefatty acids or glycerol for exercising muscles as measured by anincrease of the free fatty acids or the glycerol in blood circulatinglevels; (4) insulin secretion of no more than 5% increase followingadministration; (5) reduction of activity-induced protein catabolism;(6) increased protein biosynthesis (7) stable or increased bloodoxygenation, (8) reduced production of at least one stress hormone; (9)reduced production of protein oxidation products, (10) increased orreduced-depletion of endogenous pH buffering agents includingbeta-alanine or carnosine; and (11) reduced fatigue or reduced soreness.